a) Filed of Invention
At present, nonlinear contrasts such as two-photon absorption or second harmonic generation (SHG) are used to an increasing extent in microscopy, e.g., for examination of biological preparations. It is advantageous to use short pulse lasers to provide the energy needed to excite nonlinear effects. In this connection, the peak pulse power should be as high as possible and the pulse length at the location of the specimen should accordingly be as small as possible to prevent damage to the preparation simultaneously. Short pulse lasers supply light pulses, for example, of several 10 fs at a repetition rate of several 10 MHz. Accordingly, they have the advantage that they emit extremely high peak pulse energies accompanied at the same time by low average output.
It is disadvantageous that the short pulses on the path through the microscope to the specimen change due to the group velocity dispersion (GVD)—usually, they become longer.
b) Description of the Related Art
In order to compensate for pulse lengthening, corresponding changes (prechirp devices) have been suggested (DE 19622353). Further, adaptive optics have been provided in DE 19733193. The described devices are suitable for compensation of second-order dispersion.
However, higher-order dispersions which cannot be determined beforehand must be taken into account, e.g., in biological preparations. Further, higher-order dispersions occur in the optical components in a microscope. Therefore, it is not possible to create optimum conditions for the excitation of nonlinear contrasts by conventional techniques.
In conventional fluorescence microscopy, different dyes are used for specific tagging of biological preparations. These dyes are subsequently excited by different light wavelengths. In preparations of this kind, simultaneous excitation of the various dyes is usually carried out using multiphoton excitation. On the one hand, this is advantageous because only one light wavelength is needed for excitation. On the other hand, it is disadvantageous when the emission wavelength bands of the individual dyes overlap because the dyes can then no longer be spectrally separated.